To improve the expression level of nitrile hydratase, methods that overexpress nitrile hydratase in microorganism cells by genetic engineering and that convert nitrile compounds to the corresponding amide compounds using the cells have been examined. For example, known nitrile hydratases are derived form the following microorganisms. All of the nitrile hydratases are enzymes consisting of two types of heterogeneous subunits.
The genus Rhodococcus (Examined Published Japanese Patent Application (JP-B) NO. Hei 3-54558)
The genus Rhodococcus (Unexamined Published Japanese Patent Application (JP-A) NO. Hei 2-119778)
The genus Pseudomonas (JP-A Hei 3-251184) The genus Rhodococcus rhodochrous (EP Patent Application NO. 455646)
However, in all cases, the expression level of nitrile hydratase activity is not high enough with E. coli transformed with an expression plasmid containing an insert fragment of any nitrile hydratase gene described in these patent publications; the nitrile-hydrating activity per weight of cells of the transformant is lower than that of the original microorganism from which the gene is derived (Ikehata, O., Nishiyama, M., Horinouchi, S. and Beppu, T. “Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus speices N-774 and its expression in Escherichia coli” Eur. J. Biochem. 181(1989), 563-570; Nishiyama, M., Horinouchi, S., Kobayashi, M., Nagasawa, T., Yamada, H. and Beppu, T. “Cloning and Characterization of Genes Responsible for Metabolism of Nitrile Compounds from Pseudomonas chlororaphis B23” Journal of bacteriology 173 (1991):2465-2472; Kobayashi, M., Nishiyama, M., Nagasawa, T., Horinouchi, S., Beppu, T. and Yamada, H. “Cloning nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1” Biochimica et Biophysica Acta. 1129 (1991):23-33).
As mentioned above, it has been possible to express the nitrile hydratase activity itself in E. coli by genetic recombination techniques. However, so far, there is no available transformant having so high nitrile hydration activity as to be used for the industrial production of amides.
On the other hand, a nitrile hydratase, which exhibits very high nitrile hydration activity, has been found in Achromobacter xerosis (IFO 12668), and the gene encoding the enzyme has been cloned. Further, the gene was introduced into E. coli using an expression plasmid, and nitrile hydratase was overproduced with the resulting transformant (JP-A Hei 8-266277). This report shows only examples of acrylamide and α-hydroxyisobutylamide production, and thus there is no description on the activity of this enzyme toward 2-hydroxy-4-methylthiobutyronitrile in the report.
Thus, little is known about nitrile hydratases that can use 2-hydroxy-4-methylthiobutyronitrile (hereinafter abbreviated as HMBN) as a substrate. Furthermore, there is no previous method for producing 2-hydroxy-4-methylthiobutylamides (hereinafter abbreviated as HMBAm) from 2-hydroxy-4-methylthiobutyronitrile using E. coli transformed with an expression plasmid containing the nitrile hydratase gene as an insert fragment.
On the other hand, with respect to the production of α-hydroxyamide by microorganism, there is a known method for producing the corresponding amides from lactonitrile, hydroxyacetonitrile, α-hydroxy methylthiobutyronitrile and such, using microorganisms belonging to the genus Bacillus, the genus Bacteridium, the genus Micrococcus or the genus Brevibacterium (see JP-B Sho 62-21519). In addition, there also exists a publicly known method for producing mandelamide from cyanohydrin (see JP-A Hei 4-222591; JP-A Hei 8-89267).
However, the enzymes having the nitrile hydratase activity capable of converting nitrile compounds to amide compounds have a problem that the enzymes readily lose their own enzymatic activities due to the presence of the nitrile compound as starting material or amide compound as the product. If the concentration of nitrile compound is raised in order to increase the rate of amidation, the nitrile hydratase is readily inactivated in a short period of time, and thus it is hard to obtain amide compound as the reaction product in a desired period of time. In addition, the amide compounds as the products also readily inactivate the nitrile hydratase, and thus it is difficult to obtain a high concentration of amide compound.
Furthermore, in varying degree depending on the type of compound, α-hydroxynitrile has been known to be partially decomposed to the corresponding aldehyde and hydrocyanic acid in a polar solvent (see V. Okano et al., J. Am. Chem. Soc., Vol. 98, 4201 (1976)). In general, aldehydes are linked to proteins and can inactivate the enzymatic activity (see Chemical Modification of Proteins, G. E. Means et al., Holden-Day, 125(1971)). Further, like aldehyde, hydrocyanic acid (cyanide) can also inhibitorily act on many enzymes. Thus, aldehyde and cyanide produced from α-hydroxynitrile as the starting material can be the cause of decreased enzymatic activity. Because of a problem that the enzyme is inactivated in a short period of time in the enzymatic hydration or hydrolysis of α-hydroxy nitrile, it was difficult to obtain a high concentration of α-hydroxyamide with high productivity.
To prevent the loss of enzymatic activity, various methods for increasing the enzymatic activity or for suppressing the loss of enzymatic activity (inactivation) have been tested. Such attempts include, for example, the following:                The reaction is carried out at a lower temperature ranging from the freezing point to 15° C. (JP-B Sho 56-38118).        A lower concentration of substrate is continuously supplied from multiple supply ports (JP-B Sho 57-1234).        A microorganism or processed product thereof is treated with an organic solvent (JP-A Hei 5-308980).        The reaction is carried out in the presence of higher unsaturated fatty acid (JP-A Hei 7-265090).        The microorganism cells are subjected to crosslinking treatment with glutaraldehyde and such (JP-A Hei 7-265091; JP-A Hei 8-154691).        The concentration of hydrocyanic acid contaminated in the nitrile compound is lowered by a chemical method, and then nitrile hydratase is allowed to react with the nitrile compound (see JP-A Hei 11-123098).        The long-term stabilization of the enzymatic activity is achieved by the presence of sulfite ion, acid sulfite ion or dithionite ion (see JP-A Hei 8-89267)        Aldehyde is added (see JP-A Hei 4-222591).        
None of these methods had sufficient effects on the industrial applications. Although some of the methods were effective, they had room for economical or practical improvement. For example, the above-mentioned method adding aldehyde requires a large quantity of aldehyde in 1-5 times molar excess of cyanohydrin as the start material, and thus the method was less than an economical solution. Similarly, it is illustrated that the method adding sulfite ion, acid sulfite ion or dithionite ion requires addition of the ion in an amount equivalent to or larger than that of the starting material, and thus the method was not practical one.
An objective of the present invention is to provide a nitrile hydratase having high nitrile-hydrating activity. Another objective of the present invention is to provide a stable nitrile hydratase capable of maintaining the high enzymatic activity over a long period of time. In addition, still another important objective of the present invention is to provide a nitrile hydratase capable of also using 2-hydroxy-4-methylthiobutyronitrile as a substrate.
Furthermore, another objective of the present invention is to provide the gene encoding nitrile hydratase having high nitrile hydration activity, recombinant plasmid containing the gene and transformant containing the recombinant plasmid. In addition, yet another objective of the present invention is to provide a method for producing the corresponding amides from nitrile using the transformant expressing high nitrile hydration activity.